The long-term objective of this project is to determine the structures of all orphan nuclear receptor ligand binding domains (LBDs), to correlate their structures with the biological functions of these receptors, and to explore the structural information for discovery of drugs that target these receptors. Nuclear receptors (NRs) constitute a large family of DNA-binding transcription factors that modulate the expression of genes involved in a broad spectrum of physiology. In contrast to other transcription factors, the activity of NRs is regulated by small-molecule ligands that directly bind NRs to induce active or repressive conformations. Their regulation by small molecules has made NRs one of the most important and successful targets for therapeutic drugs. All nuclear receptors contain at least one of two highly conserved domains: the centrally located DNA-binding domain (DBD) and the C-terminal LBD. The LBD is the key structural and functional domain of a NR. In addition to ligand binding, the LBD contains dimerization motifs and a conserved surface that mediates ligand-regulated recruitment of coactivators and co-repressors for transcriptional regulation. The LBD has thus been the focus of intense structural study and the direct target of pharmaceutical discovery. Crystal structures of all classical endocrine nuclear receptors and all adopted orphan receptors (receptors for which ligands were identified after the receptor was identified) have been solved. These structures reveal a conserved sandwich fold that harbors a ligand binding pocket and a C-terminal activation helix (AF-2) that mediates the ligand-regulated function of nuclear receptors. Importantly, these structures have been instrumental in revealing key mechanisms of ligand regulation and ligand discovery for nuclear receptors, as demonstrated for SF-1 and COUP-TFII in the first period of this grant. Currently, there are only three subfamilies (SHP, GCNF, and TLX/PNR) of human nuclear receptors for which the LBD structure remains to be solved. SHP, GCNF, TLX, and PNR are essential regulators in cholesterol homeostasis, neurogenesis, eye development, and stem cell regeneration, respectively, but little is known about their structures and ligand regulation. The lack of structural information has become a critical barrier for understanding the biology and signaling pathways mediated by these receptors. Based on ligand identification for other orphan nuclear receptors and the common structural features of nuclear receptors, we hypothesize that the remaining orphan nuclear receptors (SHP, GCNF, TLX/PNR) are also ligand-regulated. In this renewed application, the plan is to use X-ray crystallography in combination with biochemical and functional assays to test this hypothesis. Achieving our specific aims will overcome the above critical barrier to scientific advancements related to these receptors by providing a comprehensive structural and molecular framework for ligand regulation of these receptors, as well as a knowledgeable foundation for drug discovery focused on targeting these receptors. PUBLIC HEALTH RELEVANCE: SHP, TLX/PNR, and GCNF are orphan nuclear receptors that play crucial roles in the metabolism of lipids and bile acid, or maintenance and development of neural and embryonic stem cells. However, little is known about their structures and aspects of ligand regulation. The lack of structural information for these receptors has become critical barriers to progress of these receptors. Crystal structures of these receptors and their functional correlation under their respective biological context will not only establish whether these orphan nuclear receptors are ligand-regulated receptors, but will also serve as a rational template for drug design targeting of these receptors for cancers, metabolic diseases, and stem cell therapy for neurodegenerative diseases.